Quinstatin compounds

ABSTRACT

The present disclosure relates to Quinstatin compounds, pharmaceutical compositions comprising such compounds, kits, and methods for using such compounds or pharmaceutical compositions.

BACKGROUND OF THE INVENTION

The discovery^(1b) of the dolastatin series^(1c,d) of cancer cell growthinhibitors contained in the wide ranging sea hare Dolabella auriculariarevealed a new and very promising vista for anticancer drug discovery.That's proved to be broadly confirmed by clinical development,² ofdolastatin 10 (1) and 15^(2c) and structural modifications designatedauristatins.³ By 2001, auristatin E (2a)^(3b) began preclinicaldevelopment as a very promising antibody drug conjugate (ADC) by linkageto a CD30 monoclonal antibody. Soon the development continued, employingthe cancer biology equivalent desmethyl auristatin E (2b).

Presently the resulting ADC anticancer drug^(4a-e) designated ADCETRIShas been approved for use in 50 countries, for example, treatment ofpatients with refractory Hodgkin's lymphoma and large cell lymphomathereby resulting in remarkable complete remissions.^(4d) Those earlyclinical results have inspired a massive current research effort to linkauristatin (2b) to other monoclonal antibodies^(4a,b) representing aspectrum of cancer types. In parallel, considerable research has beencontinued to discover structural modifications of dolastatin 10 thatwould provide powerful cancer cell growth inhibition without the hightoxic level, and provide a much better therapeutic range for inspiredADC use. Citation of any reference in this section is not to beconstrued as an admission that such reference is prior art to thepresent disclosure.

BRIEF SUMMARY OF THE INVENTION

The present disclosure relates to Quinstatin compounds, pharmaceuticalcompositions comprising such compounds, kits, and methods for using suchcompounds or pharmaceutical compositions. The compounds of the presentdisclosure contain an easily derivatizable group for conjugation tomonoclonal antibodies. The compounds have, or are believed to have,suitable cancer cell growth inhibition values.

In a first embodiment, the present disclosure provides a compound offormula (I):

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit;

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl;

R₃ is H or (C₁-C₆) alkyl; and

X is a bicyclic heterocyclic ring system selected from quinolinyl,isoquinolinyl, 1,5-naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, 2,7-naphthyridinyl, 1,8-naphthyridinyl, 2,6-naphthyridinyl,phthalazinyl, 2H-chromenyl, 1H-1,5-benzodiazepinyl, 1,2,3-benzotriazinyland 2,5-benzodiazocinyl, wherein the bicyclic heterocyclic ring systemis unsubstituted or substituted with 1, 2 or 3 substituent(s) selectedfrom (C₁-C₆) alkoxy, methylene dioxy, hydroxyl, O-Protecting Group andO-Linker Unit.

In a second embodiment, the present disclosure provides a compound offormula (Ia),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In a third embodiment, the present disclosure provides a compound offormula (Ib),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In a fourth embodiment, the present disclosure provides a compound offormula (Ic),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In a fifth embodiment, the present disclosure provides a compound offormula (Id),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and R₂ is selected from H, (C₁-C₆) alkyl,(C₂-C₆) alkenyl or (C₂-C₆) alkynyl.

In a sixth embodiment, the present disclosure provides a compound offormula (Ie),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and R₂ is selected from H, (C₁-C₆) alkyl,(C₂-C₆) alkenyl or (C₂-C₆) alkynyl.

In a seventh embodiment, the present disclosure provides a compound offormula (If),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and R₂ is selected from H, (C₁-C₆) alkyl,(C₂-C₆) alkenyl or (C₂-C₆) alkynyl.

In an eighth embodiment, the present disclosure provides a compound offormula (Ig),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

The compounds of this disclosure have or are believed to have highlyfavorable cancer cell growth inhibitory activities. In particular,compounds of formula (I) exhibit nanomolar cancer cell growth inhibitoryactivities. See Example 6. The cancer cell growth inhibitory activitiesare significantly improved relative to auristatin AQ.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes the following:

-   -   (1.) A compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit;

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl;

R₃ is H or (C₁-C₆) alkyl; and

X is a bicyclic heterocyclic ring system selected from quinolinyl,isoquinolinyl, 1,5-naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, 2,7-naphthyridinyl, 1,8-naphthyridinyl, 2,6-naphthyridinyl,phthalazinyl, 2H-chromenyl, 1H-1,5-benzodiazepinyl, 1,2,3-benzotriazinyland 2,5-benzodiazocinyl, wherein the bicyclic heterocyclic ring systemis unsubstituted or substituted with 1, 2 or 3 substituent(s) selectedfrom (C₁-C₆) alkoxy, methylene dioxy, hydroxyl, O-Protecting Group andO-Linker Unit.

-   -   (2.) The compound of the above (1.), wherein X is quinolinyl.    -   (3.) The compound of the above (1.), wherein X is isoquinolinyl.    -   (4.) The compound of the above (1.), wherein X is 2-quinolinyl,        3-quinolinyl, 6-quinolinyl, 7-quinolinyl or 8-quinolinyl.    -   (5.) The compound of the above (1.), wherein X is 2-quinolinyl,        6-quinolinyl, 7-quinolinyl or 8-quinolinyl.    -   (6.) A compound of formula (Ia),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (7.) The compound of the above (6.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (8.) The compound of the above (6.), wherein R₁ is H or (C₁-C₆)        alkyl.    -   (9.) The compound of the above (6.), wherein R₁ is H or a Linker        Unit.    -   (10.) The compound of the above (6.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (11.) The compound of the above (6.), wherein R₁ is H, methyl or        a Linker Unit.    -   (12.) The compound of the above (6.), wherein R₁ is H or methyl.    -   (13.) The compound of the above (6.), wherein R₁ is H or a        Linker Unit.    -   (14.) The compound of the above (6.), wherein R₁ is methyl or a        Linker Unit.    -   (15.) The compound of the above (6.), wherein R₁ is H.    -   (16.) The compound of the above (6.), wherein R₁ is methyl.    -   (17.) The compound of the above (6.), wherein R₁ is a Linker        Unit.    -   (18.) The compound of any of the above (6.) to (17.), wherein R₂        is H or (C₁-C₆) alkyl.    -   (19.) The compound of any of the above (6.) to (17.), wherein R₂        is H or methyl.    -   (20.) The compound of any of the above (6.) to (17.), wherein R₂        is H.    -   (21.) The compound of any of the above (6.) to (17.), wherein R₂        is (C₁-C₆) alkyl.    -   (22.) The compound of any of the above (6.) to (17.), wherein R₂        is methyl.    -   (23.) A compound of formula (Ib),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (24.) The compound of the above (23.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (25.) The compound of the above (23.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (26.) The compound of the above (23.), wherein R₁ is H or a        Linker Unit.    -   (27.) The compound of the above (23.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (28.) The compound of the above (23.), wherein R₁ is H, methyl        or a Linker Unit.    -   (29.) The compound of the above (23.), wherein R₁ is H or        methyl.    -   (30.) The compound of the above (23.), wherein R₁ is H or a        Linker Unit.    -   (31.) The compound of the above (23.), wherein R₁ is methyl or a        Linker Unit.    -   (32.) The compound of the above (23.), wherein R₁ is H.    -   (33.) The compound of the above (23.), wherein R₁ is methyl.    -   (34.) The compound of the above (23.), wherein R₁ is a Linker        Unit.    -   (35.) The compound of any of the above (23.) to (34.), wherein        R₂ is H or (C₁-C₆) alkyl.    -   (36.) The compound of any of the above (23.) to (34.), wherein        R₂ is H or methyl.    -   (37.) The compound of any of the above (23.) to (34.), wherein        R₂ is H.    -   (38.) The compound of any of the above (23.) to (34.), wherein        R₂ is (C₁-C₆) alkyl.    -   (39.) The compound of any of the above (23.) to (34.), wherein        R₂ is methyl.    -   (40.) A compound of formula (Ic),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (41.) The compound of the above (40.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (42.) The compound of the above (40.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (43.) The compound of the above (40.), wherein R₁ is H or a        Linker Unit.    -   (44.) The compound of the above (40.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (45.) The compound of the above (40.), wherein R₁ is H, methyl        or a Linker Unit.    -   (46.) The compound of the above (40.), wherein R₁ is H or        methyl.    -   (47.) The compound of the above (40.), wherein R₁ is H or a        Linker Unit.    -   (48.) The compound of the above (40.), wherein R₁ is methyl or a        Linker Unit.    -   (49.) The compound of the above (40.), wherein R₁ is H.    -   (50.) The compound of the above (40.), wherein R₁ is methyl.    -   (51.) The compound of the above (40.), wherein R₁ is a Linker        Unit.    -   (52.) The compound of any of the above (40.) to (51.), wherein        R₂ is H or (C₁-C₆) alkyl.    -   (53.) The compound of any of the above (40.) to (51.), wherein        R₂ is H or methyl.    -   (54.) The compound of any of the above (40.) to (51.), wherein        R₂ is H.    -   (55.) The compound of any of the above (40.) to (51.), wherein        R₂ is (C₁-C₆) alkyl.    -   (56.) The compound of any of the above (40.) to (51.), wherein        R₂ is methyl.    -   (57.) A compound of formula (Id),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (58.) The compound of the above (57.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (59.) The compound of the above (57.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (60.) The compound of the above (57.), wherein R₁ is H or a        Linker Unit.    -   (61.) The compound of the above (57.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (62.) The compound of the above (57.), wherein R₁ is H, methyl        or a Linker Unit.    -   (63.) The compound of the above (57.), wherein R₁ is H or        methyl.    -   (64.) The compound of the above (57.), wherein R₁ is H or a        Linker Unit.    -   (65.) The compound of the above (57.), wherein R₁ is methyl or a        Linker Unit.    -   (66.) The compound of the above (57.), wherein R₁ is H.    -   (67.) The compound of the above (57.), wherein R₁ is methyl.    -   (68.) The compound of the above (57.), wherein R₁ is a Linker        Unit.    -   (69.) The compound of any of the above (57.) to (68.), wherein        R₂ is H or (C₁-C₆) alkyl.    -   (70.) The compound of any of the above (57.) to (68.), wherein        R₂ is H or methyl.    -   (71.) The compound of any of the above (57.) to (68.), wherein        R₂ is H.    -   (72.) The compound of any of the above (57.) to (68.), wherein        R₂ is (C₁-C₆) alkyl.    -   (73.) The compound of any of the above (57.) to (68.), wherein        R₂ is methyl.    -   (74.) A compound of formula (Ie),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (75.) The compound of the above (74.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (76.) The compound of the above (74.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (77.) The compound of the above (74.), wherein R₁ is H or a        Linker Unit.    -   (78.) The compound of the above (74.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (79.) The compound of the above (74.), wherein R₁ is H, methyl        or a Linker Unit.    -   (80.) The compound of the above (74.), wherein R₁ is H or        methyl.    -   (81.) The compound of the above (74.), wherein R₁ is H or a        Linker Unit.    -   (82.) The compound of the above (74.), wherein R₁ is methyl or a        Linker Unit.    -   (83.) The compound of the above (74.), wherein R₁ is H.    -   (84.) The compound of the above (74.), wherein R₁ is methyl.    -   (85.) The compound of the above (74.), wherein R₁ is a Linker        Unit.    -   (86.) The compound of any of the above (74.) to (85.), wherein        R₂ is H or (C₁-C₆) alkyl.    -   (87.) The compound of any of the above (74.) to (85.), wherein        R₂ is H or methyl.    -   (88.) The compound of any of the above (74.) to (85.), wherein        R₂ is H.    -   (89.) The compound of any of the above (74.) to (85.), wherein        R₂ is (C₁-C₆) alkyl.    -   (90.) The compound of any of the above (74.) to (85.), wherein        R₂ is methyl.    -   (91.) A compound of formula (If),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (92.) The compound of the above (91.), wherein R₁ is H, (C₁-C₆)        alkyl or a Linker Unit.    -   (93.) The compound of the above (91.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (94.) The compound of the above (91.), wherein R₁ is H or a        Linker Unit.    -   (95.) The compound of the above (91.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (96.) The compound of the above (91.), wherein R₁ is H, methyl        or a Linker Unit.    -   (97.) The compound of the above (91.), wherein R₁ is H or        methyl.    -   (98.) The compound of the above (91.), wherein R₁ is H or a        Linker Unit.    -   (99.) The compound of the above (91.), wherein R₁ is methyl or a        Linker Unit.    -   (100.) The compound of the above (91.), wherein R₁ is H.    -   (101.) The compound of the above (91.), wherein R₁ is methyl.    -   (102.) The compound of the above (91.), wherein R₁ is a Linker        Unit.    -   (103.) The compound of any of the above (91.) to 102.), wherein        R₂ is H or (C₁-C₆) alkyl.    -   (104.) The compound of any of the above (91.) to (102.), wherein        R₂ is H or methyl.    -   (105.) The compound of any of the above (91.) to (102.), wherein        R₂ is H.    -   (106.) The compound of any of the above (91.) to (102.), wherein        R₂ is (C₁-C₆) alkyl.    -   (107.) The compound of any of the above (91.) to (102.), wherein        R₂ is methyl.    -   (108.) A compound of formula (Ig),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

-   -   (109.) The compound of the above (108.), wherein R₁ is H,        (C₁-C₆) alkyl or a Linker Unit.    -   (110.) The compound of the above (108.), wherein R₁ is H or        (C₁-C₆) alkyl.    -   (111.) The compound of the above (108.), wherein R₁ is H or a        Linker Unit.    -   (112.) The compound of the above (108.), wherein R₁ is (C₁-C₆)        alkyl or a Linker Unit.    -   (113.) The compound of the above (108.), wherein R₁ is H, methyl        or a Linker Unit.    -   (114.) The compound of the above (108.), wherein R₁ is H or        methyl.    -   (115.) The compound of the above (108.), wherein R₁ is H or a        Linker Unit.    -   (116.) The compound of the above (108.), wherein R₁ is methyl or        a Linker Unit.    -   (117.) The compound of the above (108.), wherein R₁ is H.    -   (118.) The compound of the above (108.), wherein R₁ is methyl.    -   (119.) The compound of the above (108.), wherein R₁ is a Linker        Unit.    -   (120.) The compound of any of the above (108.) to (119.),        wherein R₂ is H or (C₁-C₆) alkyl.    -   (121.) The compound of any of the above (108.) to (119.),        wherein R₂ is H or methyl.    -   (122.) The compound of any of the above (108.) to (119.),        wherein R₂ is H.    -   (123.) The compound of any of the above (108.) to (119.),        wherein R₂ is (C₁-C₆) alkyl.    -   (124.) The compound of any of the above (108.) to (119.),        wherein R₂ is methyl.    -   (125.) The compound of any of the above (1.) to (124.), wherein        the Linker Unit comprises a cleavable linker.    -   (126.) The compound of the above (125.), wherein the cleavable        linker is cleavable by a method selected from the group        consisting of glycosidase-induced cleavage, acid-induced        cleavage, light-induced cleavage, peptidase-induced cleavage,        esterase-induced cleavage, and disulfide bond cleavage.    -   (127.) The compound of the above (126.), wherein the cleavable        linker comprises a glycosidic bond, a hydrazone, a        cathepsin-B-cleavable peptide, a disulfide or an ester bond.    -   (128.) The compound of any one of claims 1 to 124, wherein the        Linker Unit has formula:        A_(a)W_(w)Y_(y), wherein

A_(a) is maleimidocaproyl, W_(w) is Valine-Citrulline and Y_(y) isp-aminobenzyloxycarbonyl.

-   -   (129.) The compound of the above (126.), wherein the cleavable        linker comprises glucuronide.    -   (130.) The compound of any of the above (1) to (124) or (128.),        wherein the Linker Unit comprises a monoclonal antibody.    -   (131.) A compound selected from the group consisting of:

and

pharmaceutically acceptable salts thereof.

-   -   (132.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (133.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (134.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (135.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (136.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (137.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (138.) A compound, which is

and a pharmaceutically acceptable salt thereof.

-   -   (139.) A pharmaceutical composition comprising a combination of        compounds of the above (1.) to (138.) or pharmaceutically        acceptable salts thereof and a pharmaceutically acceptable        carrier.    -   (140.) The pharmaceutical composition of the above (139.) or the        above (140.), further comprising a therapeutically effective        amount of a second therapeutic agent selected from the group        consisting of a tubulin-forming inhibitor, a topoisomerase        inhibitor, and a DNA binder.    -   (141.) A method for killing or inhibiting the proliferation of        tumor cells or cancer cells comprising treating the tumor cells        or cancer cells with a compound of any of the above (1.) to        (138.), or a pharmaceutical composition of any one of the above        (139.) to (141.), in an amount effective to kill or inhibit the        proliferation of the tumor cells or cancer cells.    -   (142.) A method for treating cancer in a patient in need thereof        comprising administering to the patient a compound of any of the        above (1.) to (138.), or a pharmaceutical composition of any one        of the above (139.) to (141.), wherein the compound or        pharmaceutical composition is administered in an amount        effective to treat cancer.    -   (143.) The method of the above (143.), further comprising        administering an effective amount of a second therapeutic agent.    -   (144.) The method of the above (143.), wherein the cancer is        selected from the group consisting of breast cancer, ovarian        cancer, stomach cancer, endometrial cancer, salivary gland        cancer, lung cancer, kidney cancer, colon cancer, colorectal        cancer, thyroid cancer, pancreatic cancer, prostate cancer,        bladder cancer and central nervous system cancer.    -   (145.) The method of the above (143.), wherein the cancer is        selected from the group consisting of breast cancer, lung        cancer, colon cancer, pancreatic cancer, prostate cancer and        central nervous system cancer.    -   (146.) A method of determining inhibition of cellular        proliferation by a compound, comprising contacting cells in a        cell culture medium with the compound of any of the above (1.)        to (138.) and measuring the cytotoxic activity of the compound,        whereby proliferation of the cells is inhibited.    -   (147.) A method of inhibiting the growth of tumor cells that        overexpress a tumor-associated antigen comprising administering        to a patient the compound of any of the above (1.) to (138.)        conjugated to an antibody that is specific for said        tumor-associated antigen, and optionally a second therapeutic        agent wherein the compound and the second therapeutic agent are        each administered in amounts effective to inhibit the growth of        tumor cells in the patient.    -   (148.) The method of the above (148.), wherein the compound        sensitizes the tumor cells to said second therapeutic agent.    -   (149.) The method of the above (148.), wherein the compound        induces cell death.    -   (150.) The method of the above (148.), wherein the compound        induces apoptosis.    -   (151.) A use of the compound of any of the above (1.) to (138.)        in the manufacture of a medicament for treating cancer.    -   (152.) An article of manufacture comprising the compound of any        of the above (1.) to (138.), a container, and a package insert        or label indicating that the compound can be used to treat        cancer characterized by the overexpression of at least one        tumor-associated antigen.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. Allpublications, patents and other documents mentioned herein areincorporated by reference in their entirety.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

The term “a” or “an” may mean more than one of an item.

The terms “and” and “or” may refer to either the conjunctive ordisjunctive and mean “and/or”.

The term “about” means within plus or minus 10% of a stated value. Forexample, “about 100” would refer to any number between 90 and 110.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe disclosure. Exemplary salts include, but are not limited to,sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

The term “pharmaceutically acceptable carrier” refers to a diluent,adjuvant or excipient, with which a compound of the disclosure may beadministered. Pharmaceutically acceptable carriers include any and allsolvents, diluents, or other liquid vehicles, dispersions or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. Remington's PharmaceuticalSciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton,Pa., 1980) discloses various carriers used in formulatingpharmaceutically acceptable compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with the compounds of the disclosure such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticallyacceptable composition, its use is contemplated to be within the scopeof this disclosure. Examples of pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols, such apropylene glycol or polyethylene glycol; esters, such as ethyl oleateand ethyl laurate; agar; buffering agents such as magnesium hydroxideand aluminum hydroxide; alginic acid; pyrogen-free water; isotonicsaline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

The term “therapeutically effective amount” refers to an amount of acompound of the disclosure or a pharmaceutically acceptable salt thereofeffective to treat a cancer in a patient. For purposes of thisdisclosure, the therapeutically effective amount of the compound mayreduce the number of cancer cells; reduce the tumor size; reduce to someextent cancer cell infiltration into peripheral organs, tumor metastasisor tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer.

The terms “treat” or “treatment” refer to therapeutic treatment andprophylactic measures to obtain a beneficial or desired result. Forpurposes of this disclosure, beneficial or desired results include, butare not limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), whether detectableor undetectable and prevention of relapse. “Treatment” can also includeprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already having thecondition or disorder as well as those prone to have the condition ordisorder.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells.

Exemplary cancers include, but not limited to, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, central nervous system cancer, squamouscell carcinoma, basal cell carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, small cell lung carcinoma,bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma multiforme, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, retinoblastoma, acute lymphoblastic leukemia (ALL), acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL),acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, multiple myeloma, Hodgkin's disease, non-Hodgkin'slymphoma, Waldenstrom's macroglobulinemia, heavy chain disease andpolycythemia vera.

The term “cytotoxic activity” refers to a cell-killing, a cytostatic oran anti-proliferative effect of a compound of the disclosure. Methodsfor measuring cytotoxic activity are well-known in the art.

Cytotoxic activity may be expressed as the IC₅₀ value, which is theconcentration (molar or mass) per unit volume at which half the cellssurvive.

The term “patient,” as used herein, includes, but is not limited to, ahuman, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat,bird and fowl. In some embodiments, the patient is a human.

The term “(C₁-C₆) alkyl” refers to saturated linear or branchedhydrocarbon structures having 1, 2, 3, 4, 5, or 6 carbon atoms. When analkyl residue having a specific number of carbons is named, allgeometric isomers having that number of carbons are intended to beencompassed; thus, for example, “propyl” includes n-propyl andiso-propyl and “butyl” includes n-butyl, sec-butyl, iso-butyl andtert-butyl. Examples of “(C₁-C₆) alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, and thelike.

The term “(C₁-C₃) alkyl” refers to saturated linear or branchedhydrocarbon structures having 1, 2 or 3 carbon atoms. When an alkylresidue having a specific number of carbons is named, all geometricisomers having that number of carbons are intended to be encompassed;thus, for example, “propyl” includes n-propyl and iso-propyl. Examplesof “(C₁-C₃) alkyl groups include methyl, ethyl, n-propyl and iso-propyl.

The term “(C₂-C₆) alkenyl” refers to a straight-chain or branchedunsaturated hydrocarbon radical having 2, 3, 4, 5 or 6 carbon atoms anda double bond in any position, e.g., ethenyl, 1 propenyl, 2 propenyl(allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-methylethenyl, 1-methyl-1propenyl, 2 methyl-2-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 2-methyl-2-pentenyl,4-methyl-2-pentenyl, 4-methyl-1-pentenyl, 3-methyl-1-pentenyl, and thelike.

The term “(C₂-C₆)alkynyl” refers to a straight chain or branchedhydrocarbon having 2, 3, 4, 5 or 6 carbon atoms and including at leastone carbon-carbon triple bond. Examples of alkynyl include ethynyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 1-hexynyl,2-hexynyl, 3-hexynyl, 4-methyl-2-pentynyl and the like.

The term “Protecting Group” refers to any group that is capable ofreversibly protecting another functional group from undergoing anundesired reaction. Suitable oxygen and nitrogen protecting groups, aswell as suitable conditions for protection and deprotection arewell-known in the art and are described e.g., in T. W. Greene and P. G.M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley,New York 1999, and references cited therein. Representative hydroxyprotecting groups include acetates (e.g., pivaloate and benzoate),benzyl ether, p-methoxybenzyl ether, trityl ether, tetrahydropyranylether, trialkylsilyl ethers (e.g., trimethylsilyl ether, triethylsilylether, triisopropyl silyl ether, t-butyldimethyl silyl ether,triphenylmethyl silyl ether), allyl ethers, methoxymethyl ether,2-methoxyethoxymethyl ether, methanesulfonate and p-toluenesulfonate.Representative amino protecting groups include, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC),and the like.

The term “antibody” as used herein includes whole antibodies, monoclonalantibodies, polyclonal antibodies, monospecific antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that exhibit the desired biological activity. An antibody maybe of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody may bederived from any suitable species. In some embodiments, the antibody isof human or murine origin.

An antibody may be, for example, human, humanized or chimeric.

The term “monoclonal antibodies” as used herein refers to antibodiesproduced by a single clone of cells or cell line and comprisingidentical antibody molecules. The term “polyclonal antibodies” refers toantibodies produced by more than one type of cell or cell line andcomprising different antibody molecules.

A compound of the disclosure can contain one, two, or more asymmetriccenters and thus can give rise to enantiomers, diastereomers, and otherstereoisomeric forms. The disclosure encompasses compounds with all suchpossible forms, as well as their racemic and resolved forms or anymixture thereof, unless specifically otherwise indicated. When acompound of the disclosure contains an olefinic double bond, a C═Ndouble bond, or any other center of geometric asymmetry, it is intendedto include all “geometric isomers”, e.g., both Z and E geometricisomers, unless specifically otherwise indicated. All “tautomers”, e.g.,amine-imine, enamine-enimine, enamine-imine, urea-isourea, ketone-enol,amide-imidic acid, lactam-lactim, are intended to be encompassed by thedisclosure as well unless specifically otherwise indicated.

Compounds of Formula (I)

In one embodiment, the present disclosure provides a compound of formula(I),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit;

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl;

R₃ is H or (C₁-C₆) alkyl; and

X is a bicyclic heterocyclic ring system selected from quinolinyl,isoquinolinyl, 1,5-naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, 2,7-naphthyridinyl, 1,8-naphthyridinyl, 2,6-naphthyridinyl,phthalazinyl, 2H-chromenyl, 1H-1,5-benzodiazepinyl, 1,2,3-benzotriazinyland 2,5-benzodiazocinyl, wherein the bicyclic heterocyclic ring systemis unsubstituted or substituted with 1, 2 or 3 substituent(s) selectedfrom (C₁-C₆) alkoxy, methylene dioxy, hydroxyl, O-Protecting Group andO-Linker Unit.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₃ is H or (C₁-C₆) alkyl. In another embodiment, R₃is H or methyl. In another embodiment, R₃ is H. In another embodiment,R₃ is (C₁-C₃) alkyl. In another embodiment, R₃ is methyl.

In one embodiment, X is quinolinyl or isoquinolinyl. In anotherembodiment, X is quinolinyl. In another embodiment, X is isoquinolinyl.In another embodiment, X is 1,5-naphthyridinyl. In another embodiment, Xis quinoxalinyl. In another embodiment, X is quinazolinyl. In anotherembodiment, X is cinnolinyl. In another embodiment, X is2,7-naphthyridinyl. In another embodiment, X is 1,8-naphthyridinyl. Inanother embodiment, X is 2,6-naphthyridinyl. In another embodiment, X isphthalazinyl. In another embodiment, X is 2H-chromenyl. In anotherembodiment, X is 1H-1,5-benzodiazepinyl. In another embodiment, X is1,2,3-benzotriazinyl. In another embodiment, X is 2,5-benzodiazocinyl.In each embodiment, X can be attached to the rest of the molecule at anyavailable position on the bicyclic heterocyclic ring system.

In one embodiment, X is 2-quinolinyl, 3-quinolinyl, 4-quinolinyl,5-quinolinyl, 6-quinolinyl, 7-quinolinyl or 8-quinolinyl. In anotherembodiment, X is 2-quinolinyl, 3-quinolinyl, 6-quinolinyl, 7-quinolinylor 8-quinolinyl. In another embodiment, X is 2-quinolinyl, 3-quinolinyl,6-quinolinyl, 7-quinolinyl or 8-quinolinyl. In another embodiment, X is2-quinolinyl, 6-quinolinyl, 7-quinolinyl or 8-quinolinyl. In anotherembodiment, X is 2-quinolinyl. In another embodiment, X is 3-quinolinyl.In another embodiment, X is 4-quinolinyl. In another embodiment, X is5-quinolinyl. In another embodiment, X is 6-quinolinyl. In anotherembodiment, X is 7-quinolinyl. In another embodiment, X is 8-quinolinyl.

In one embodiment, X is 1-isoquinolinyl, 3-isoquinolinyl,4-isoquinolinyl, 5-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl or8-isoquinolinyl. In another embodiment, X is 1-isoquinolinyl,3-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl or 8-isoquinolinyl. Inanother embodiment, X is 1-isoquinolinyl, 3-isoquinolinyl,6-isoquinolinyl, 7-isoquinolinyl or 8-isoquinolinyl. In anotherembodiment, X is 1-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl or8-isoquinolinyl. In another embodiment, X is 1-isoquinolinyl. In anotherembodiment, X is 3-isoquinolinyl. In another embodiment, X is4-isoquinolinyl. In another embodiment, X is 5-isoquinolinyl. In anotherembodiment, X is 6-isoquinolinyl. In another embodiment, X is7-isoquinolinyl. In another embodiment, X is 8-isoquinolinyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₃ is Hor (C₁-C₃) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₃is H or (C₁-C₃) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₃ is H or (C₁-C₃) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₃ is H or (C₁-C₃) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₃ is H or (C₁-C₃) alkyl. In anotherembodiment, R₁ is H or methyl and R₃ is H or (C₁-C₃) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₃ is H or (C₁-C₃)alkyl. In another embodiment, R₁ is H and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₁ is methyl and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₃ is H or (C₁-C₃) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₃ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₃ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₃ is (C₁-C₃) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₃ is methyl.

In one embodiment, R₁ is methyl and R₃ is methyl. In another embodiment,R₁ is H and R₃ is methyl. In another embodiment, R₁ is H and R₃ is H. Inanother embodiment, R₁ is a Linker Unit and R₃ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₃ is H.

In one embodiment, R₂ is H or (C₁-C₆) alkyl and R₃ is H or (C₁-C₃)alkyl. In another embodiment, R₂ is H and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₂ is (C₁-C₆) alkyl and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₂ is H or methyl and R₃ is H or (C₁-C₃) alkyl. Inanother embodiment, R₂ is methyl and R₃ is H or (C₁-C₃) alkyl.

In one embodiment, R₂ is H or (C₁-C₆) alkyl and R₃ is H. In anotherembodiment, R₂ is H or (C₁-C₆) alkyl and R₃ is methyl. In anotherembodiment, R₂ is H and R₃ is H. In another embodiment, R₂ is (C₁-C₆)alkyl and R₃ is H. In another embodiment, R₂ is methyl and R₃ is H. Inanother embodiment, R₂ is H and R₃ is (C₁-C₃) alkyl. In anotherembodiment, R₂ is H and R₃ is methyl. In another embodiment, R₂ is(C₁-C₆) alkyl and R₃ is (C₁-C₃) alkyl. In another embodiment, R₂ is(C₁-C₆) alkyl and R₃ is methyl. In another embodiment, R₂ is methyl andR₃ is methyl.

In one embodiment, the compound of formula (I) may be conjugated to anantibody. The antibody may be conjugated to the compound of the formula(I) through the N-terminus or the C-terminus. In one embodiment, theantibody is conjugated to the antibody at the N-terminus. In anotherembodiment, the antibody is conjugated to the antibody at theC-terminus. In one embodiment, the compound of formula (I) is conjugateddirectly to an antibody. In another embodiment, the compound of formula(I) is conjugated to an antibody through a Linker Unit. The Linker Unitcan operate to provide a suitable release of the compound of formula(I). The preparation of antibody drug conjugates is known to those ofskill in the art.

In embodiments in which the compound of formula (I) is conjugated to anantibody through a Linker Unit, the Linker Unit may comprise a cleavablelinker in one embodiment and a non-cleavable linker in anotherembodiment.

In embodiments in which R₁ comprises a cleavable linker, the cleavablelinker may be cleaved by methods known in the art. In one embodiment,the cleavable linker may be cleaved by a method selected from the groupconsisting of glycosidase-induced cleavage, acid-induced cleavage,light-induced cleavage, peptidase-induced cleavage, esterase-inducedcleavage, and disulfide bond cleavage. In one embodiment, the cleavagemethod is selected from the group consisting of glycosidase-inducedcleavage, acid-induced cleavage, peptidase-induced cleavage,esterase-induced cleavage, and disulfide bond cleavage. In anotherembodiment, the cleavage method is selected from the group consisting ofglycosidase-induced cleavage, peptidase-induced cleavage, andesterase-induced cleavage. In another embodiment, the cleavage method isselected from glycosidase-induced cleavage or peptidase-inducedcleavage. In another embodiment, the cleavage method is selected fromglycosidase-induced cleavage or esterase-induced cleavage. In anotherembodiment, the cleavage method is selected from peptidase-inducedcleavage or esterase-induced cleavage.

In embodiments in which R₁ comprises a cleavable linker, the cleavablelinker may comprise a glycosidic bond, a hydrazone, acathepsin-B-cleavable peptide, a disulfide or an ester bond. In oneembodiment, the cleavable linker comprises a glycosidic bond, ahydrazone, a cathepsin-B-cleavable peptide, or an ester bond. In oneembodiment, the cleavable linker comprises a glycosidic bond, ahydrazone, or a cathepsin-B-cleavable peptide. In one embodiment, thecleavable linker comprises a glycosidic bond, a hydrazone, or an esterbond. In one embodiment, the cleavable linker comprises a glycosidicbond, a cathepsin-B-cleavable peptide, or an ester bond. In oneembodiment, the cleavable linker comprises a hydrazone, acathepsin-B-cleavable peptide, or an ester bond.

In one embodiment, the cleavable linker comprises a glycosidic bond. Inone embodiment, the cleavable linker comprises glucuronide.

The compounds of the disclosure may be conjugated to any antibody, e.g.,an antibody that binds to a tumor associated antigen. In one embodiment,the antibody used in the antibody drug conjugate of the disclosure is amonoclonal antibody. The antibody can be a chimeric antibody, ahumanized antibody or an antibody fragment. In another embodiment, theantibody used in the antibody drug conjugate of the disclosure binds atleast one of CD19, CD20, CD30, CD33, CD70, BCMA, Glypican-3, Liv-1 andLewis Y antigens.

In one embodiment, the Linker Unit is a bifunctional moiety that can beused to conjugate a compound of formula (I) to an antibody. Suchbifunctional moieties are known in the art and include, but are notlimited to, alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:repeating units of alkyloxy (e.g., polyethylenoxy, PEG,polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine™);and diacid ester and amides including succinate, succinamide,diglycolate, malonate, and caproamide. See, e.g., U.S. Pat. Nos.6,214,345 and 7,745,394, the contents of both of which are incorporatedby reference in their entireties.

In one embodiment, the Linker Unit is as described in U.S. Pat. Nos.6,214,345 and 7,745,394 and has formula:A_(a)W_(w)Y_(y),

wherein A is a Stretcher Unit,

a is 0 or 1,

each —W— is independently an Amino Acid Unit,

w is an integer ranging from 0 to 12,

Y is a Spacer Unit, and

y is 0, 1 or 2.

The Stretcher Unit (-A-), when present, is capable of linking anantibody to the Amino Acid Unit (—W—). The antibody has a functionalgroup that can form a bond with a functional group of a Stretcher.Useful functional groups that can be present on an antibody, eithernaturally or via chemical manipulation include, but are not limited to,sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl groupof a carbohydrate, and carboxyl. In one aspect, the antibody functionalgroups are sulfhydryl and amino. Sulfhydryl groups can be generated byreduction of an intramolecular disulfide bond of an antibody.Alternatively, sulfhydryl groups can be generated by reaction of anamino group of a lysine moiety of an antibody using 2-iminothiolane(Traut's reagent) or another sulfhydryl generating reagent.

The Amino Acid Unit (—W—), when present, links the Stretcher Unit to theSpacer Unit if the Spacer Unit is present, links the Stretcher Unit tothe compound of formula (I) if the Spacer Unit is absent, and links theantibody to the compound of formula (I) if the Stretcher Unit and SpacerUnit are absent.

W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. The Amino Acid may be any aminoacid. In some embodiments, the Amino Acid Unit comprises natural aminoacids. In other embodiments, the Amino Acid Unit comprises non-naturalamino acids.

The Spacer Unit (—Y—), when present, links an Amino Acid Unit to thecompound of formula (I) when an Amino Acid Unit is present. Alternately,the Spacer Unit links the Stretcher Unit to the compound of formula (I)when the Amino Acid Unit is absent. The Spacer Unit also links thecompound of formula (I) to the antibody when both the Amino Acid Unitand Stretcher Unit are absent.

Suitable Spacer Units include, but are not limited to a glycine-glycineunit; a glycine unit; p-aminobenzyl alcohol (PAB) unit or aromaticcompounds that are electronically similar to the PAB group such as2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med.Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals; spacers thatundergo cyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al., ChemistryBiology, 1995, 2, 223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972,94, 5815) and 2-aminophenylpropionic acid amides (Amsberry, et al., J.Org. Chem., 1990, 55, 5867); and a branched bis(hydroxymethyl)styrene(BHMS) unit.

Examples of Linker Units, Stretching Units and Amino Acid Units aredescribed in U.S. Pat. Nos. 6,214,345 and 7,745,394.

In some embodiments, A_(a) is maleimidocaproyl (mc).

In some embodiments, W_(w) is Valine-Citrulline (ValCit).

In some embodiments, Y_(y) is p-aminobenzyloxycarbonyl (PABC).

In some embodiments, A_(a) is maleimidocaproyl, W, is Valine-Citrullineand Y_(y) is p-aminobenzyloxycarbonyl (mcValCitPABC).

In some embodiments, the Linker Unit is selected from maleimidocaproyl;mcValCitPABC-, MalPegXC2-, AmPegXC2-, mcValCitPABCAmPegXC2-,MalPegXC2ValCitPABC-, 2BrAcPegXC2, my-, mb-me-, MalC6-,PFPCOPe2XC2ValCitPABC-, PFPCOPegXC2AmPegYC2-, PFPCOPegXC2AlaAlaAsnPABC-,PFPCOPegXC2-, PFPCOPegXC2AmPegYC2PABC-, mcGly-, AzCOC2Ph4AmCOPeg2C2-,AzCOC2Ph4AmPeglC1-, and AcLysValCitPABC-, each of which is described inU.S. Pat. No. 9,249,186.

The term “MalPegXC2-” refers to

The term “AmPegXC2-” refers to

The term “mcValCitPABCAmPegXC2-” refers to

The term “MalPegXC2ValCitPABC-” refers to

The term “2BrAcPegXC2” refers to

The term “mv-” refers to

The term “mb” refers to

The term “me-” refers to

The term “MalC6-” refers to

The term “PFPCOPe2XC2ValCitPABC-” refers to

The term “PFPCOPegXC2AmPegYC2-” refers to

The term “PFPCOPegXC2AlaAlaAsnPABC-” refers to

The term “PFPCOPegXC2-” refers to

The term “PFPCOPegXC2AmPegYC2PABC-” refers to

The term “mcGly-” refers to

The term “AzCOC2Ph4AmCOPeg2C2-” refers to

The term “AzCOC2Ph4AmPeglC1-” refers to

The term “AcLysValCitPABC-” refers to

In another embodiment, the present disclosure provides a compound offormula (Ia),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Ia) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Ia) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Ia) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (Ib),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Ib) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Ib) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Ib) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (Ic),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Ic) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Ic) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Ic) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (Id),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Id) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Id) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Id) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (Ie),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Ie) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Ie) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Ie) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (If),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (If) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (If) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (If) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

In another embodiment, the present disclosure provides a compound offormula (Ig),

or a pharmaceutically acceptable salt thereof, wherein

R₁ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl,a Protecting Group or a Linker Unit; and

R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl or (C₂-C₆)alkynyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit. In anotherembodiment, R₁ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is (C₁-C₆) alkyl or a LinkerUnit. In another embodiment, R₁ is H, methyl or a Linker Unit. Inanother embodiment, R₁ is H or methyl. In another embodiment, R₁ is H ora Linker Unit. In another embodiment, R₁ is methyl or a Linker Unit. Inanother embodiment, R₁ is H. In another embodiment, R₁ is methyl. Inanother embodiment, R₁ is a Linker Unit.

In one embodiment, R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₂is H or methyl. In another embodiment, R₂ is H. In another embodiment,R₂ is (C₁-C₆) alkyl. In another embodiment, R₂ is methyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor (C₁-C₆) alkyl. In another embodiment, R₁ is H or (C₁-C₆) alkyl and R₂is H or (C₁-C₆) alkyl. In another embodiment, R₁ is H or a Linker Unitand R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁ is (C₁-C₆) alkylor a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In another embodiment, R₁is H, methyl or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or methyl and R₂ is H or (C₁-C₆) alkyl. In anotherembodiment, R₁ is H or a Linker Unit and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl or a Linker Unit and R₂ is H or (C₁-C₆)alkyl. In another embodiment, R₁ is H and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is methyl and R₂ is H or (C₁-C₆) alkyl. Inanother embodiment, R₁ is a Linker Unit and R₂ is H or (C₁-C₆) alkyl.

In one embodiment, R₁ is H, (C₁-C₆) alkyl or a Linker Unit and R₂ is Hor methyl. In another embodiment, R₁ is H, (C₁-C₆) alkyl or a LinkerUnit and R₂ is H. In another embodiment, R₁ is H, (C₁-C₆) alkyl or aLinker Unit and R₂ is (C₁-C₆) alkyl. In another embodiment, R₁ is H,(C₁-C₆) alkyl or a Linker Unit and R₂ is methyl.

In one embodiment, R₁ is methyl and R₂ is methyl. In another embodiment,R₁ is H and R₂ is methyl. In another embodiment, R₁ is H and R₂ is H. Inanother embodiment, R₁ is a Linker Unit and R₂ is methyl. In anotherembodiment, R₁ is a Linker Unit and R₂ is H.

In one embodiment, the compound of formula (Ig) may be conjugated to anantibody, e.g., selected from the antibodies described above inconnection with the compound of formula (I). The antibody may beconjugated to the compound of formula (Ig) in the manner described abovein connection with the compound of formula (I).

In embodiments in which the compound of formula (Ig) is conjugated to anantibody through a Linker Unit, the Linker Unit may be selected from theLinker Units described above in connection with the compound of formula(I).

Representative compounds of formula (I) include:

and pharmaceutically acceptable salts thereof.

In one embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound is

or a pharmaceutically acceptable salt thereof.

Additional representative compounds of formula (I) include:

and pharmaceutically acceptable salts thereof, wherein

R₄ is a Linker Unit having formula:A_(a)W_(w)Y_(y),

A_(a) is maleimidocaproyl, W_(w) is Valine-Citrulline and Y_(y) isp-aminobenzyloxycarbonyl.

Pharmaceutical Compositions

According to another embodiment, the present disclosure provides apharmaceutical composition comprising a compound described herein or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

The pharmaceutical composition of the disclosure may be formulated foradministration in solid or liquid form, including those adapted foradministration by oral, nasal, parenteral, rectal, topical, ocular,inhalation and intra-tumor administration. Parenteral administrationincludes subcutaneous injections, intravenous, intramuscular orintrasternal injection or infusion techniques. In one embodiment, thecompositions are administered parenterally. In another embodiment, thecompositions are administered intravenously.

The pharmaceutical composition of the disclosure may be in the form of aliquid, e.g., a solution, emulsion or suspension, pellets, powders,sustained-release formulations, or any other form suitable for use. Thepharmaceutical composition may comprise sterile diluents such as water,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono- ordigylcerides, which can serve as the solvent or suspending medium,polyethylene glycols, glycerin, cyclodextrin, propylene glycol or othersolvents; antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates, phosphates or amino acids; agents for the adjustment oftonicity such as sodium chloride or dextrose; surfactants;preservatives; wetting agents; dispersing agents; suspending agents;stabilizers; solubilizing agents; local anesthetics, e.g., lignocaine;or isotonic agent.

It should be understood that a specific dosage and treatment regimen forany particular patient will depend upon a variety of factors, includingthe type of patient (e.g., human), the activity of the specific compoundemployed, the composition employed, the manner of administration, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the nature and the severity of the particular disorderbeing treated. The amount of active ingredients will also depend uponthe particular compound in the composition. The amount of activeingredient can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays can optionally be employed to helpidentify optimal dosage ranges.

Preferably, the compositions are formulated so that a dosage of betweenabout 0.01 to about 20 mg/kg body weight/day of the compound of formula(I) can be administered to a patient receiving the composition. In oneembodiment, the dosage administered to the patient is between about 0.01mg/kg and about 10 mg/kg of the patient's body weight. In anotherembodiment, the dosage administered to the patient is between about 0.1mg/kg and about 10 mg/kg of the patient's body weight. In yet anotherembodiment, the dosage administered to the patient is between about 0.1mg/kg and about 5 mg/kg of the patient's body weight. In yet anotherembodiment, the dosage administered is between about 0.1 mg/kg and about3 mg/kg of the patient's body weight. In yet another embodiment, thedosage administered is between about 1 mg/kg and about 3 mg/kg of thepatient's body weight.

The pharmaceutical compositions comprise an effective amount of acompound described herein such that a suitable dosage will be obtained.Typically, this amount is at least about 0.01% of a compound by weightof the composition. In a preferred embodiment, pharmaceuticalcompositions are prepared so that a parenteral dosage unit contains fromabout 0.01% to about 2% by weight of the compound of the disclosure.

For intravenous administration, the pharmaceutical composition maycomprise from about 0.01 to about 100 mg of a compound described hereinper kg of the patient's body weight. In one aspect, the composition mayinclude from about 1 to about 100 mg of a compound described herein perkg of the patient's body weight. In another aspect, the amountadministered will be in the range from about 0.1 to about 25 mg of acompound described herein per kg of body weight.

The pharmaceutical compositions of the disclosure may optionally furthercomprise a second therapeutic agent in a therapeutically effectiveamount. The second therapeutic agent includes those that are known andthose discovered to be effective in treating cancer. In someembodiments, the second therapeutic agent may be selected from the groupconsisting of a tubulin-forming inhibitor, a topoisomerase inhibitor,and a DNA binder.

Methods of Use

According to another embodiment, the present disclosure provides methodsof using the compounds described herein or pharmaceutical compositionsthereof. The compounds and compositions are useful for killing orinhibiting the proliferation of tumor cells or cancer cells. Thecompounds and compositions are also useful for treating cancer in apatient.

In some embodiments, the present disclosure provides methods of killingor inhibiting the proliferation of tumor cells or cancer cells. In someembodiments, the method comprises contacting the tumor cells or cancercells with a compound described herein, or a pharmaceutically acceptablesalt thereof, in an amount effective to kill or inhibit theproliferation of the tumor cells or cancer cells. In alternateembodiments, the method comprises contacting the tumor cells or cancercells with a pharmaceutical composition comprising a compound of formula(I) in an amount effective to kill or inhibit the proliferation of thetumor cells or cancer cells.

In some embodiments, the method further comprises contacting the cellswith an effective amount of a second therapeutic agent or apharmaceutical composition thereof. In one embodiment, the secondtherapeutic agent is selected from the group consisting of atubulin-forming inhibitor, a topoisomerase inhibitor, and a DNA binder.

The cells may be contacted with the compound described herein and thesecond therapeutic agent simultaneously in either the same or differentcompositions or sequentially in any order. The amounts of compounddescribed herein and the second therapeutic agent and the relativetimings of their contact will be selected in order to achieve thedesired combined effect.

In another embodiment, the present disclosure provides a method ofdetermining inhibition of cellular proliferation by a compound describedherein. The method comprises contacting cells in a cell culture mediumwith the compound described herein and measuring the cytotoxic activityof the compound, whereby proliferation of the cells is inhibited. Insome embodiments, the method further comprises culturing the cells for aperiod from about 6 hours to about 5 days.

Suitable cell lines are known to those skilled in the art and includethose used for evaluating other anti-cancer drugs. Such cell linesinclude, but are not limited to, BXPC-3 (pancreas); MCF-7 (breast);SF-268 (CNS); NCI-H460 (lung); KM20L2 (colon); DU-145 (prostate); 786-0,(renal cell carcinoma); Caki-1 (renal cell carcinoma); L428 (Hodgkin'sdisease); UMRC-3 (renal cell carcinoma); LP-1 (human myeloma); and U251(glioblastoma). In some embodiments, the cells are obtained from apatient having a disease to be treated (e.g., cancer) or from a relevantcell line.

In another embodiment, the present disclosure provides a method ofmeasuring cell viability in the presence of a compound described herein.The method comprises contacting cells in a cell culture medium with thecompound of described herein, culturing the cells for a period fromabout 6 hours to about 5 days, preferably 96 hours; and measuring cellviability. In some embodiments, the cells are obtained from a patienthaving a disease to be treated (e.g., cancer) or from a relevant cellline.

In another embodiment, the present disclosure provides a method fortreating cancer in a patient. In some embodiments, the method comprisesadministering to the patient a compound described herein, or apharmaceutically acceptable salt thereof, in an amount effective totreat cancer. In other embodiments, the method comprises administeringto the patient a composition comprising a compound described herein inan amount effective to treat cancer.

In some embodiments, the patient receives an additional treatment, suchas radiation therapy, surgery, and chemotherapy with anotherchemotherapeutic agent or combinations thereof. In some embodiments, thecompound of the disclosure is administered concurrently with thechemotherapeutic agent or with radiation therapy or with surgery. Inother embodiments, the chemotherapeutic agent or radiation therapy orsurgery is administered or performed prior or subsequent toadministration of a compound of the disclosure.

In some embodiments, the method for treating cancer further comprisesadministering to the patient an effective amount of a second therapeuticagent, e.g., a chemotherapeutic agent. Any one or a combination of thechemotherapeutic agents, such a standard of care chemotherapeuticagent(s), can be administered.

In some embodiments, the chemotherapeutic agent may be selected from thegroup consisting of a tubulin-forming inhibitor, a topoisomeraseinhibitor, and a DNA binder.

The compound described herein and the chemotherapeutic agent may beadministered simultaneously in either the same or differentpharmaceutical composition or sequentially in any order. The amounts ofcompound described herein and the chemotherapeutic agent and therelative timings of their administration will be selected in order toachieve the desired combined effect.

In another embodiment, the present disclosure provides a method ofinhibiting the growth of tumor cells that overexpress a tumor-associatedantigen in a patient. In some embodiments, the method comprisesadministering to the patient a compound described herein conjugated toan antibody that is specific for said tumor-associated antigen, whereinthe compound described herein is administered in amount effective toinhibit growth of tumor cells in the patient. In alternate embodiments,the method comprises administering to the patient a pharmaceuticalcomposition comprising a compound described herein conjugated to anantibody that is specific for said tumor-associated antigen, wherein thecompound described herein is administered in amount effective to inhibitgrowth of tumor cells in the patient. The method may optionally furthercomprises administering to the patient a chemotherapeutic agent, or apharmaceutical composition thereof, in an amount effective to inhibitthe growth of tumor cells in the patient.

In some embodiments, the compound sensitizes the tumor cells to thechemotherapeutic agent.

In some embodiments, the compound induces cell death. In otherembodiments, the compound induces apoptosis.

In some embodiments, the tumor cells are associated with a cancerselected from the group consisting of breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, colorectal, thyroid,pancreatic, prostate, central nervous system and bladder cancer.

In some embodiments, the compound described herein is conjugated to anantibody selected from the group consisting of CD19, CD20, CD30, CD33,CD70, BCMA, Glypican-3, Liv-1 and Lewis Y.

Any compound or pharmaceutical composition described herein may be usedin the methods of the present disclosure.

In some of the above methods, the compound described herein isadministered to a patient in a composition comprising a pharmaceuticallyacceptable carrier. In some of these embodiments, the composition isadministered intravenously. In certain embodiments, the compound isformulated in a unit dosage injectable form.

In preferred embodiments of each of the above methods, the patient is ahuman.

In an additional embodiment, the present disclosure provides the use ofa compound of described herein in the manufacture of a medicament forthe treatment of any of the above mentioned cancers. It will beappreciated that a compound described herein and one or morechemotherapeutic agents may be used in the manufacture of themedicament.

In additional embodiments, the present disclosure provides an article ofmanufacture comprising a compound described herein, a container, and apackage insert or label indicating that the compound can be used totreat cancer characterized by the overexpression of at least onetumor-associated antigen.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indication(s), usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLES

The compounds of this disclosure may be prepared by methods known tothose skilled in the art, or the methods set forth below. By followingthe methods below, additional compounds of the disclosure can beprepared by modifying the choice of starting materials, reagents andreaction conditions as known to those skilled in the art.

General Experimental Procedures

Both N-Boc-Dolaproine and Dov-Val-Dil.TFA were synthesized as describedearlier.^(6,7) 2-(1′-amino-2′-ethyl)-quinoline dihydrochloride and6-quinoline acetic acid were purchased from J&W Pharmlab LLC andAstatech, Inc respectively, and were used as received. 3-quinolineacetic acid and 7-quinoline acetic acid were purchased from PrincetonBimolecular Research, Inc. and 8-(1′-amino-2′-ethyl)-quinoline waspurchased from Fisher Scientific and all used as received.4-(1′-amino-2′-ethyl)-quinoline dihydrochloride and5-(1′-amino-2′-ethyl)-quinoline dihydrochloride were purchased fromEnamine Ltd. Other reagents and anhydrous solvents were purchased fromAcros Organics (Fisher Scientific), Sigma-Aldrich Chemical Company andwere used as received. For thin-layer chromatography, Analtech silicagel GHLF Uniplates were used and visualized with short-wave UVirradiation and an iodine chamber. For column chromatography, silica gel(230-400 mesh ASTM) from E. Merck (Darmstadt, Germany) was used. Meltingpoints are uncorrected and were determined with a Fischer-Johns meltingpoint apparatus.

Optical rotations were measured by use of a Perkin-Elmer 241polarimeter, and the [α]_(D) values are given in 10⁻¹ deg cm² g⁻¹. The¹H, ¹³C spectra were recorded on Varian Unity INOVA 400 and 500instruments with deuterated solvents. High-resolution mass spectra wereobtained with a Jeol JMS-LCmate mass spectrometer.

Example 1. Synthesis of Quinstatin 2

Scheme 1 depicts the synthesis of Quinstatin 2 (3).

2-(1′-Boc-Dap-amino-2′-ethyl)-quinoline (5)

To a stirred solution of Boc-Dap⁶ (0.06 g, 0.21 mmol) and2-(1′-amino-2′-ethyl)-quinoline dihydrochloride (0.08 g, 0.32 mmol) inanhydrous DCM (1 mL) at 0° C. under N₂ was added TEA (0.2 mL, 1.42 mmol)and diethylcyanophosphonate (DECP) (0.1 mL, 0.6 mmol). The reactionmixture was stirred at 0° C. with warming to room temperature for 6 h,and then concentrated under reduced pressure to an orange coloredresidue which was purified by chromatography on a silica gel column.Gradient elution with hexanes 100% to 7:2 hexanes—acetone gave theproduct as a yellow residue under reduced pressure. The product wasfurther separated on a silica gel column, and eluting with 7:2hexanes:acetone gave an off-white waxy solid, 112 mg (79%); TLC R_(f)0.5 (3:4 hexanes-acetone); Doubling of signals observed in the ¹H and¹³C NMR data indicating conformational isomers in almost 1:1 ratio dueto cis-trans isomerism at the Dap bond⁷; ¹H NMR (CDCl₃, 400 MHz) δ 8.01(1H, d, J=8 Hz), 7.95 (1H, d, J=8 Hz), 7.72 (1H, d, J=8 Hz), 7.63 (1H,t, J=8 HZ), 7.43 (1H, t, J=8 Hz), 7.23 (1H, d, J=8 Hz), 6.99, 6.85 (1H,brs, 2×1H), 3.83-3.56 (4H, m), 3.46-3.34 (1H, m), 3.28 (4H, m),3.18-3.01 (m, 2H), 2.36-2.15 (m, 1H), 1.83-1.47 (m, 4H), 1.37, 1.41 (s,9H, t-Bu), 1.12-1.09 (m, 3H). ¹³C NMR (CDCl₃, 400 MHz) (two conformerswere observed) 174.1, 173.6, 160.1 154.6, 154.3, 147.6, 136.5, 129.6,128.7, 127.6, 126.8, 126.1, 121.7, 83.9, 82.3, 79.6, 79.0, 60.7, 60.5,58.7, 46.8, 46.5, 44.2, 43.9, 38.3, 38.1, 37.5, 37.3, 28.5, 25.7, 25.3,252, 24.4, 23.9, 14.1, 14.05. HRAPCIMS m/z 442.2707 [M+H]⁺ (calcd forC₂₅H₃₆N₃O₄ 442.2706).

2-(1′-Boc-Dap-amino-2′-ethyl)-quinoline trifluoroacetate (6)

To a stirred solution of compound 5 (0.070 g, 0.16 mmol) in DCM (4 mL)at 0° C. under N₂ was added trifluoroacetic acid (TFA) (1 mL) andstirring was continued for 1.5 h at 0° C. The reaction was stopped andconcentrated under reduced pressure to remove DCM and excess TFA toyield a yellow oil. This material was used without further purificationin the next reaction.

2-(1′-Dov-Val-Dil-Dap-2′-ethyl)-quinoline (3, Quinstatin 2)

Amide 6 and Dov-Val-Dil.TFA⁷ (90 mg, 0.16 mmol) were dissolved inanhydrous DCM (5 mL) and the solution was stirred under N₂ and cooled to0° C. TEA (0.12 mL, 0.86 mmol) and DECP (0.035 mL, 0.21 mmol) wereadded, and the mixture was stirred under N₂ for 18 h with warming toroom temperature. The reaction mixture was concentrated under reducedpressure and separated on a silica gel column. Elution with DCM:MeOH(95:5) gave the product as a colorless foam: yield 74 mg (62%); TLCR_(f) 0.3 (CH₂Cl₂:CH₃OH 7%); [α]₂₂ ^(D)—8.2 (c 0.27, CHCl₃);Conformational isomers are present^(3a) (˜2:1) and doubling of signalswere observed in both the proton and carbon NMR spectra for 3; ¹H NMR(CDCl₃, 400 MHz) major conformer δ 8.08 (1H, d, J=8.8 Hz), 8.02 (1H, d,J=8.4 Hz), 7.79 (1H, d, J=7.0 Hz), 7.70 (1H, t, J=8.0 Hz), 7.51 (1H, t,J=6.8 Hz), 7.33 (1H, d, J=9.0 Hz), 7.16 (1H, m), 6.88 (1H, m), 4.80 (1H,d, J=6.9 Hz), 4.78 (1H, d, J=6.9 Hz), 4.12-4.04 (2H, m), 3.89-3.74 (3H,m), 3.33 (3H, s), 3.27 (3H, s), 3.21 (1H, t, J=6.8 Hz), 3.17 (1H, s),3.01 (3H, s), 2.48-2.29 (3H, m), 2.25 (6H, s), 2.13-1.84 (7H, m), 1.73(1H, m), 1.43-1.32 (1H, m), 1.25-1.20 (m, 3H), 1.06-0.8 (21H, m); ¹³CNMR (CDCl₃, 400 MHz) two conformers, 174.0, 173.5, 173.3, 171.6, 170.1,169.9, 160.3, 159.9, 147.7, 147.6, 36.7, 136.3, 129.7, 129.4, 128.8,128.7, 127.6, 1275, 126.8, 126.8, 126.2, 125.9, 121.7, 121.6, 86.0,82.0, 78.2, 76.5, 76.4, 61.5, 60.3, 59.0, 58.1, 57.9, 53.73, 53.68,53.5, 47.5, 46.5, 44.9, 44.0, 42.8, 38.5, 37.9, 37.8, 37.4, 33.1, 30.9,27.6, 25.9, 25.7, 24.9, 24.7, 23.4, 20.1, 17.8, 17.7, 15.8, 15.2, 14.3,10.7, 10.3 ppm. HRAPCIMS m/z 753.5292 [M+H]+(calcd for C₄₂H₆₉N₆O₆,753.5279).

Example 2. Synthesis of Quinstatin 3 3-(1′-Hydroxyl-2′-ethyl)-quinoline

To a stirred solution containing 3-quinoline acetic acid (0.03 g, 0.16mmol) in anhydrous tetrahydrofuran (THF) (2 mL) at 0° C. was addeddropwise LiAlH₄ (1 M solution in THF, 0.2 mL, 0.3 mmol, 1.25 equiv.).

The reaction mixture was stirred at 0° C. for 1 h, dampened with thesuccessive addition of water (8 μL), 15% NaOH (8 μL) and again water (24μL), prior to being stirred for 45 min at 0-5° C. and drying (Na₂SO₄).The solution was filtered to remove the precipitated solids and thefiltrate was concentrated under reduced pressure to give a yellow oilwhich solidified on standing to a waxy solid (15.8 mg, 57%); TLC R 0.17(DCM-MeOH 3%); ¹H NMR (CDCl₃, 400 MHz) δ 8.71 (1H, s), 8.01 (1H, d,J=7.5 Hz), 7.96 (1H, s), 7.70 (1H, d, J=8.4 Hz), 7.62 (1H, t, J=7.5 Hz),7.48 (1H, t, J=7.5 Hz), 3.97 (2H, t, J=6.4 Hz), 3.01 (2H, t, J=6.4 Hz);13C NMR (CDCl₃, 100 MHz) 151.9, 146.6, 135.5, 131.8, 128.9, 128.8,128.0, 127.4, 126.7, 62.9, 36.5.

3-(1′-Bromo-2′-ethyl)quinoline

A stirred solution of 3-(1′-Hydroxyl-2′-ethyl)-quinoline (0.15 g, 0.86mmol) in anhydrous benzene (20.0 mL) were gently heated to effectsolution and cooled to 0° C. Phosphorous tribromide (0.2 ml, 2.4 g, 2.1mmol, 2.4 equiv.) in benzene (1.5 mL) was added and the mixture heatedto 60° C. for 45 min, then cooled to 0° C. Saturated aq. NaHCO₃ (6 mL)was added until neutral pH. The mixture was extracted with ethyl acetate(3×10 ml) and the combined organic extracts were washed with water,dried (Na₂SO₄) and concentrated to give a yellow residue which wasseparated using silica gel column chromatography (sgc) and a gradientelution of CH₂Cl₂ 100%→CH₂Cl₂—CH₃OH 4%, to yield the product as a yellowoil (68 mg, 34% yield); R_(f)=0.54 (CH₂Cl₂:CH₃OH 4%); ¹H NMR (CDCl₃, 400MHz) δ 8.79 (1H, s), 8.09 (1H, d, J=8.8 Hz), 8.00 (1H, s), 7.80 (1H, d,J=8.0 Hz) 7.63 (1H, t, J=7.8 Hz) 7.54 (1H, t, J=8 Hz), 3.66 (2H, t,J=7.8 Hz), 3.36 (2H, t, J=7.2 Hz). ¹³C NMR (CDCl₃, 400 MHz) 151.6 (×2),135.4, 131.7, 129.5, 129.4, 128.1, 127.7, 127.1, 36.6, 32.5 PPM.

3-(1′-azido-2′-ethyl)quinoline

To a stirred solution containing 3-(1′-Bromo-2′-ethyl)quinoline (0.023g, 0.1 mmol) in anhydrous DMF (0.5 mL) was added NaN₃ (15 mg, 0.23 mmol,2.3 equiv.). The reaction mixture was heated to 90° C. for 30 min,cooled and diluted with ethyl acetate (3 mL), washed with water (3 mL),brine (3 mL), dried (MgSO₄), and concentrated to give the azide as abrown oil, 15.5 mg, 79% yield; R_(f)=0.50 (CH₂Cl₂:CH₃OH 3%); ¹H NMR(CDCl₃, 400 MHz) δ 8.79 (1H, s) 8.13 (1H, d, J=8 Hz), 8.07 (1H, d, J=8Hz), 7.67 (1H, s), 7.59 (1H, d, J=8.8 Hz), 7.41 (1H, dd, J=8, 4 Hz),3.63 (2H, t, J=7 Hz), 3.09 (1H, t, J=7 Hz); 13C NMR (CDCl₃, 100 Hz)150.2, 147.4, 137.8, 136.4, 135.7, 130.6, 129.8, 127.1, 121.3, 52.2,35.3 PPM.

3-(1′-amine-2′-ethyl)quinoline

To a stirred solution of the preceding azide (0.015 g, 0.075 mmol) inanhydrous THF (1 mL) cooled to 0° C. under N₂, was added dropwise LiAlH₄(1M solution in THF, 0.1 mL, 0.1 mmol, 1.3 equiv.). Stirring at 0° C.was continued for 45 min. The reaction was completed by the dropwiseaddition of H₂O (4 μL), 15% NaOH (4 μL), and H₂O (12 μL) successively,and stirring was continued for 30 min. The mixture was diluted withethyl acetate (5 mL) dried (Na₂SO₄), and the product solution filtered.The filter cake was washed with ethyl acetate (5 mL), the organic layerscombined, concentrated and dried under reduced pressure to give a yellowoil (11 mg, 84%); ¹H NMR (CDCl₃, 400 MHz) indicated a mixture of thedesired amine along with a dihydroquinoline based on the observedupfield shift of the aromatic ring signals in the proton spectrum. Thematerial was carried forward as this mixture to coupling with Boc-Dap.

3-(1′-Boc-Dap-2′-ethyl)-quinoline

To a solution of the preceding amine mixture (0.04 g, 0.23 mmol) inanhydrous DCM (1 mL) was added a solution of Boc-Dap⁶ (0.06 g, 0.22mmol) in anhydrous DCM (1 mL) and the resulting mixture cooled to 0° C.Triethylamine (0.2 ml, 1.43 mmol) and DECP (0.12 mL, 0.8 mmol) wereadded and the solution stirred at 0° C. with warming to room temperaturefor 6 h. Concentration under reduced pressure led to a dark orange oilwhich was separated by sgc. Elution with a gradient 100% Hexane-1: 1-2:3hexanes-acetone yielded a dark orange oil, 100 mg. Purification wasachieved using flash sg chromatography and a gradient elution of Hexane100%→Hexane-Acetone 1:4. The early fractions were combined to give anoil (56 mg) which on 1H NMR analysis was found to be Boc-dap startingmaterial contaminated with DEPC reagent. The product was obtained as afoam (20 mg, 21% yield); ¹H NMR (CDCl₃, 400 MHz): δ 8.77 (1H, s), 8.06(1H, d, J=8.6 Hz), 7.98 (1H, bs), 7.74 (1H, d, J=8 Hz), 7.66 (1H, t,J=7.44 Hz), 7.52 (1H, t, J=7.44 Hz), 6.59 (1H, bs), 5.85 (1H, bs),3.81-3.41 (4H, m), 3.34 (3H, s), 3.20-3.3.08 (2H, m), 3.06-2.99 (2H, m),2.28 (1H, m), 1.83-1.70 (2H, m), 1.64-1.54 (2H, m), 1.50-1.38 (9H, m),1.22-1.13 (3H, m). (+)HRAPCIMS m/z 442.2707 [M+H]⁺ (calcd for C₂₅H₃₆N₃O₄442.2706).

3-(1′-Dap-amino-2′-ethyl)-quinoline Trifluoroacetate salt

To a stirred solution of amide (0.02 g, 0.04 mmol) in anhydrous DCM (1.5mL) at 0° C. under N₂ was added TFA (0.3 mL, 0.2 g, 1.76 mmol, 44equiv.). The solution was stirred for 1.5 h at 0° C., and thenconcentrated and dried under reduced pressure to give a residue whichwas used immediately in the next reaction.3-(1′-Dov-Val-Dil-Dap-2′-ethyl)-quinoline (Quinstatin 3). To a stirredsolution of the preceding TFA salt (0.015 g, 0.04 mmol) and Dov-Val-DilTFA⁷ (0.025 g, 0.04 mmol) in anhydrous DCM (3 mL) at 0° C. under N₂ wasadded TEA (0.03 mL) followed by DECP (0.011 mL) and the solution stirredat 0° C. for 3 h. The solvent was removed at room temperature underreduced pressure to give a yellow oil which was separated bychromatography on a silica gel column eluting with a gradient ofDCM-MeOH 4% to DCM-MeOH 12% which gave the product in the finalfractions as an oil, 30 mg, containing contaminants which were removedby extracting the product into dichloromethane and washing with water,dried (Na₂SO₄), and concentrated to an off-white glass solid (powderwhen scratched) 10 mg (29% yield); TLC R_(f)=0.14 (Hexane-Acetone 1:1);¹H NMR (CDCl₃, 400 MHz) δ 8.75 (1H, bs), 8.03 (1H, d, J=8 Hz), 7.98 (1H,s), 7.74 (1H, d, J=8 Hz), 7.63 (1H, t, J=8 Hz), 7.50 (1H, t, J=7.6 Hz),6.90 (1H, bs), 4.73 (1H, t, J=8 Hz), 3.95 (1H, m), 3.84 (1H, m), 3.78(1H, d, J=9 Hz), 3.75-3.47 (2H, m), 3.38-3.18 (2H, m), 3.38 (3H, s),3.21 (3H, s), 3.14-3.0 (3H, m), 2.97 (3H, s), 2.64-2.47 (6H, bs), 2.28(2H, m), 2.17-1.79 (3H, m), 1.64 (2H, m), 1.35-1.11 (6H, m), 1.07-0.74(21H, m); (+)HRAPCIMS m/z 753.5282 [M+H]⁺ (calcd for C₄₂H₆₉N₆O₆,753.5279).

Example 3. Synthesis of Quinstatin 6

Scheme 2 depicts the Synthesis of Quinstatin 6 (4).

2-(Quinolin-6-yl)ethan-1-ol (7)

2-(Quinolin-6-yl)Acetic acid (0.09 g, 0.48 mmol) in anhydrous THF (9 mL)was stirred at 0° C. (ice bath). A 1M solution of LiAlH₄ in THF (0.6 mL,0.6 mmol, 1.25 equiv.) was added dropwise.⁸ The reaction mixture wasthen stirred for 1 h at 0° C. and terminated with the addition of H₂O(0.02 mL), 15% NaOH (0.02 mL) and H₂O (0.06 mL). The product mixture wasstirred for 45 min at 0° C. then dried (Na₂SO₄). The solids were removedby filtration and the organic filtrate was concentrated and driedfurther under reduced pressure to alcohol 7, a yellow oil, 72 mg, 86%yield. ¹H NMR (CDCl₃, 400 MHz) δ 8.82 (dd, 1H, J=1.6, 4.3 Hz), 8.08 (d,1H, J=8 Hz), 8.00 (d, 1H, J=8 Hz), 7.64 (bs, 1H), 7.58 (dd, 1H, J=8, 1.8Hz), 7.36 (dd, 1H, J=8.8, 4.4 Hz), 3.98 (t, 2H, J=6.5 Hz), 3.06 (t, 1H,J=6.5 Hz). ¹³C-NMR (CDCl₃, 100 MHz) 150.3, 147.4, 137.5, 135.9, 131.3,129.7, 127.4, 125.7, 121.4, 63.4, 39.4 PPM.

6-(2-Bromoethyl)quinoline (8):⁹

To a stirred solution of alcohol 7 (0.12 g, 0.67 mmol) in anhydrousbenzene (2.0 mL) at 0° C. was added a solution of phosphorous tribromide(0.9 ml, 5.6 mmol, 8 equiv.) in benzene (1.0 mL). The mixture was heatedto 60° C. for 45 min, then cooled to room temperature and sat. aq.NaHCO₃ (100 mL) was added until a neutral pH was achieved. The mixturewas then extracted with ethyl acetate (3×20 ml) and the combined organicextracts were washed with water, dried (Na₂SO₄) and concentrated to givethe bromide as a yellow oil, 0.12 mg, 76% yield. ¹H NMR (CDCl₃, 400 MHz)δ 8.90 (s, 1H), 8.13 (d, 1H, J=8.4 Hz), 8.08 (d, 1H, J=8.4 Hz), 7.66 (s,1H), 7.58 (d, 1H, J=8.4 Hz), 7.39 (m, 1H), 3.65 (t, 2H, J=7.2 Hz), 3.34(t, 1H, J=7.2 Hz). ¹³C NMR (CDCl₃, 400 MHz) 150.2, 147.4, 137.2,135.7×2, 130.5, 129.8, 127.1, 121.3, 39.1, 32.5 PPM.

6-(2-Azidoethyl)quinoline (9)

To a stirred solution of bromide 8 (0.12 g, 0.5 mmol) in anhydrous DMF(3 mL) was added NaN₃ (120 mg, 1.84 mmol, 3.7 equiv.) and the reactionmixture heated to 90° C. for 30 min. and after cooling the reactionmixture, it was diluted with ethyl acetate (30 mL), washed with water(30 mL), followed by brine (30 mL), dried (MgSO₄) and concentrated togive the azide as a brown oil, 91 mg, 91% yield. ¹H NMR (CDCl₃, 400 MHz)δ 8.91 (s, 1H), 8.13 (d, 1H, 8 Hz), 8.07 (d, 1H, J=8 Hz), 7.67 (s, 1H),7.59 (d, 1H, J=8.8 Hz), 7.41 (dd, 1H, J=8, 4 Hz), 3.63 (t, 2H, J=7 Hz),3.09 (t, 1H, J=7 Hz). ¹³C-NMR (CDCl₃, 100 Hz) 150.2, 147.4, 137.8,136.4, 135.7, 130.6, 129.8, 127.1, 121.3, 52.2, 35.3 PPM.

2-(Quinolin-6-yl)ethan-1-amine (10)

To a stirred solution of azide 9 (0.089 g, 0.45 mmol) in anhydrous THF(3 mL) cooled to 0° C. under N₂ was added dropwise a 1M solution ofLiAlH₄ in THF (0.75 mL, 0.75 mmol, 1.65 equiv.). Stirring at 0° C. wascontinued for 45 min. The reaction was completed by the dropwiseaddition of H₂O (30 μL), 15% NaOH (30 μL), and H₂O (90 μL) successivelyand stirring was continued for 20 min. The mixture was diluted withethyl acetate (10 mL) dried (Na₂SO₄), and the solution filtered. Thefilter cake was washed with ethyl acetate (5 mL), the organic layerscombined, concentrated and dried under reduced pressure to give amine 8as a yellow oil (quantitative yield). ¹H NMR (CDCl₃, 500 MHz) 8.88 (m,1H), 8.11 (d, 1H, J=8.3 Hz), 8.06 (d, 1H, J=8.7 Hz), 7.63 (s, 1H), 7.59(d, 1H, J=8.5 Hz), 7.39 (dd, 1H, J=8, 4 Hz), 3.09 (t, 2H, J=7 Hz), 2.95(t, 2H, J=7 Hz), 1.81 (bs, 2H) PPM. ¹³C-NMR (CDCl₃, 120 Hz) 149.9,147.3, 137.6, 135.6, 131.0, 129.6, 126.9, 121.2, 43.2, 39.9 PPM.

Boc-Dap-2(quinolin-6-yl)ethylamine) (11):³c

To a solution of the amine 10 (0.083 g, 0.5 mmol) in anhydrous DCM (3mL) was added a solution of Boc-Dap (0.153 g, 0.5 mmol) in anhydrous DCM(1 mL) and the resulting mixture cooled to 0° C. Triethylamine (0.3 ml,2.1 mmol) and DECP (0.2 mL, 1.2 mmol) were added and the solutionstirred at 0° C. for 2 h, then concentrated under reduced pressure to adark orange oil which was separated on a silica gel column. Elution witha gradient 3:2-1:1-*2:3 of hexanes:acetone gave the desired amide as acolorless oil: 111 mg, 54% yield; TLC R_(f)0.5 (hexanes:acetone 1:1); HNMR (CDCl₃, 400 MHz) δ 8.88 (s, 1H), 8.10-8.04 (m, 2H), 7.65-7.58 (m,2H), 7.39 (m, 1H), 6.42, 5.73 (bs, NH), 3.85-3.42 (m, 4H), 3.35 (s, 3H),3.37-3.27 (m, 1H), 3.19-3.10 (m, 1H), 3.09-3.01 (m, 2H), 2.40-2.21 (m,1H), 1.84-1.71 (m, 3H), 1.53-1.42 (m, 10H), 1.19 (nm, 3H) ppm. HRMS APCI(+) m/z 442.2695 (M+H)⁺ (100), (calcd. for C₂₅H36N₃O₄, 442.2706).

Dap-2-(quinolin-6-yl)ethylamine Trifluoroacetate (12)

To a stirred solution of amine 11 (0.1 g, 0.23 mmol) in anhydrous DCM at0° C. under N₂ was added TFA (1 mL). The solution was stirred for 2 h at0° C., and then concentrated under reduced pressure to give a residuewhich was taken up in toluene and reconcentrated (×2). The oily TFA saltwas dried under reduced pressure to a consistent weight (2 h, 96 mg) andused immediately in the next reaction.

Dov-Val-Dil-Dap-2-(quinolin-6-yl)ethylamine (Quinstatin 6) (4)

To a stirred solution of TFA salt 13 (0.96 mg) and Dov-Val-Dil TFA(0.114 g, 0.21 mmol) in anhydrous. DCM (3 mL) at 0° C. under N₂ wasadded TEA (0.12 mL, 0.86 mmol) followed by DECP (0.035 mL, 0.2 mmol) andthe solution stirred at 0° C. for 2 h. The solvent was removed at roomtemperature under reduced pressure to give a yellow oil which wasseparated by chromatography on a short silica gel column (7×3 cm) andeluting slowly with hexanes:acetone 1:1 to give Quinstatin 6 as a yellowfoamy solid, 78 mg (47%); TLC R_(f)0.23 (DCM:MeOH 6%); [α]₂₂ ^(D)—6.5 (c0.34, CHCl₃) H NMR (CDCl₃, 400 MHz) 8.88 (dd, 1H, J=4.4, 1.6 Hz), 8.10(d, 1H, J=8 Hz), 8.03 (d, 1H, J=8.8 Hz), 7.66 (s, 1H), 7.61 (dd, 1H,J=8, 2 Hz), 7.39 (dd, 1H, J=8.0, 4.0 Hz), 6.88 (d, 1H, J=10 Hz), 6.72(broad t, 1H, J=4.4 Hz), 4.79 (dd, 1H, J=10, 6.8 Hz), 4.09-4.03 (m, 1H),3.98 (m, 1H), 3.82 (dd, 1H, J=8.8 Hz, 1.6 Hz), 3.71 (m, 1H), 3.59 (m,1H), 3.43-3.35 (m, 2H), 3.33 (s, 3H), 3.28 (s, 3H), 3.07 (t, 2H, J=8Hz), 3.03 (s, 3H), 2.49-2.43 (m, 1H), 2.40-2.31 (m, 2H), 2.28-2.24 (m,7H), 2.14-1.84 (m, 5H), 1.66 (m, 2H), 1.39-1.26 (m, 2H), 1.24-1.14 (m,4H), 1.06-0.92 (m, 15H), 0.85-0.82 (m, 4H) ppm. ¹³C-NMR (CDCl₃, 400 MHz)174.5, 172.0, 170.7, 170.3, 150.2, 147.6, 138.1, 135.9, 131.4, 129.7,128.6, 127.3, 121.5, 81.9, 78.8, 76.7, 60.9, 59.7, 58.2, 54.1, 47.9,44.8, 43.1, 40.7, 37.6, 35.6, 33.3, 31.3, 27.9, 26.0, 25.1, 20.1, 19.8,18.4, 18.1, 16.1, 15.3, 10.9 ppm. HRMS (APCI⁺) m/z 753.5279 [M+H]⁺(calcd for C₄₂H₆₉N₆O₆, 753.5279).

Example 4. Quinstatin7

The general experimental procedure summarized above for the synthesis ofthe 1′-ethylamine intermediate from the corresponding 3 and 6-quinolineacetic acids was employed for preparation of the amine intermediaterequired for the synthesis of Quinstatin 7.

7-(1′-hydroxyl-2′-ethyl)-quinoline

Yellow oil which solidified on standing to a waxy solid, yield 86%; TLCR_(f) 0.25 (CH₂Cl₂—CH₃OH 3%); ¹H NMR (CDCl₃, 400 MHz) δ 8.81 (1H, dd,J=4.6, 1.7 Hz), 8.09 (1H, d, J=8.5 Hz), 7.93 (1H, s), 7.73 (1H, d, J=8.8Hz), 7.42 (1H, dd, J=8.8, 0.16 Hz), 7.32 (1H, dd, J=8.5, 4.6 Hz), 3.99(2H, t, J=6.0 Hz), 3.07 (2H, t, J=6.0 Hz); ¹³C NMR (CDCl₃, 100 MHz)150.3, 140.7, 135.9 (×2C), 128.4, 127.8, 126.9, 120.6, 63.2, 39.4.

7-(1′-Bromo-2′-ethyl)-quinoline

Colorless oil (66% yield); TLC R_(f)=0.6 DCM-MeOH 3%; ¹H NMR (CDCl₃ 400MHz) δ 8.89 (1H, dd, J=4.0, 1.2 Hz), 8.81 (1H, d, J=8.7 Hz), 7.98 (1H,s), 7.79 (1H, d, J=8.7 Hz), 7.41 (2H, m), 3.65 (2H, t, J=7.9 Hz), 3.36(2H, t, J=7.6 Hz). 13C NMR (CDCl₃, 400 MHz) 149.9, 147.3, 141.1, 136.8,128.1, 128.06, 127.8, 127.2, 120.9, 39.2, 32.2 PPM.

7-(1′-azido-2′-ethyl)-quinoline

Brown oil, quantitative yield; ¹H NMR (CDCl₃, 400 MHz) 8.88 (1H, dd,J=4.0, 1.6 Hz), 8.11 (1H, d, J=8 Hz), 7.93 (1H, s), 7.76 (1H, d, J=8Hz), 7.40 (1H, dd, J=8.8, 2 Hz), 7.35 (1H, dd, J=8, 4 Hz), 3.61 (2H, t,J=7.4 Hz), 3.09 (2H, t, J=7.1 Hz); 13C NMR (CDCl₃, 100 MHz) 150.9,148.5, 139.9, 136.0, 128.9, 128.3, 128.0, 127.3, 121.1, 52.3, 35.7 PPM.7-(1′-amino-2′-ethyl)-quinoline. Yellow residue, (91% yield); ¹H NMR(CDCl₃, 400 MHz) δ 8.86 (1H, dd, J=4.4, 1.8 Hz), 8.11 (1H, dd, J=8, 1.2Hz), 7.9 (1H, s), 7.74 (1H, d, J=8 Hz), 7.39 (1H, dd, J=8, 1.5 Hz), 7.34(1H, dd, J=8, 4.4 Hz), 3.08 (2H, t, J=6.8 Hz), 2.96 (2H, t, J=6.8 Hz),1.63 (bs, NH2).

7-(1′-Boc-Dap-amino-2′-ethyl)-quinoline

To a solution of the preceding amine (0.06 g, 0.35 mmol) in anhydrousDCM was added a solution of Boc-Dap⁶ (0.1 g, 0.35 mmol) in anhydrous DCM(1 mL) and the resulting mixture cooled 0° C. Triethylamine (0.2 mL,1.43 mmol, 4 equiv.) and DEPC (0.185 mL, 0.199 g, 1.22 mmol, 3.5 equiv.)were added. The mixture was stirred at 0° C. for 8 h, then overnight atroom temperature for 24 h. The reaction mixture was next concentrated toa dark brown solid, and extracted with water, dried (Na₂SO₄) andconcentrated. Purification on sg flash using DCM-MeOH 5% gave theproduct as a yellow oil, 65 mg (43% yield based on Boc-Dap); TLCR_(f)0.4 (DCM-MeOH 5%); ¹H NMR (CDCl₃, 400 MHz) δ 8.82 (1H, bs), 8.08(1H, bd, J=9 Hz), 7.87 (1H, s), 7.71 (1H, d, J=8 Hz), 7.39 (1H, m), 7.31(1H, m), 6.51, 5.99 (1H, bs, NH), 3.77 (1H, m), 3.70 (1H, m), 3.66-3.52(3H, m), 3.43-3.35 (1H, m), 3.29 (3H, s) 3.16-2.96 (2H, m), 2.33-2.15(1H, m), 1.84-1.64 (2H, m), 1.63-1.51 (2H, m), 1.43, 1.39 (9H, s),1.16-1.08 (3H, m); (+)HRAPCIMS m/z 442.2696 [M+H]⁺ (calcd forC₂₅H₃₆N₃O₄, 442.2706).

7-(1′-Boc-Dap-amino-2′-ethyl)-quinoline Trifluoroacetate salt

A solution of the preceding ethylamide (0.06 g, 0.136 mmol) in dry DCMwas stirred and cooled to 0° C. under N₂. TFA (0.15 mL) was added andthe solution stirred at 0° C. for 3 h. The solvent was removed underreduced pressure using toluene as an azeotrope, to yield a residue whichwas further dried under high vacuum for 16 h. The salt was used as is inthe next step.

7-(1′-Dov-Val-Dil-Dap-2′-ethyl)-quinoline (Quinstatin 7)

The preceding TFA salt (0.06 g) and Dov-Val-Dil-TFA⁷ (0.078 g, 0.143mmol, 1 equiv.) were taken up in anhydrous DCM and stirred at 0° C. TEA(0.1 mL, 0.72 mmol, 5 equiv.) and DEPC (0.022 mL, 1.43 mmol) were addedin succession and the solution was stirred under argon for 4 h at 0° C.The solvent was removed under reduced pressure and the residuechromatographed using flash sg, eluting with DCM-MeOH 5%; Column size:20 cm×2 cm; 25 g silica gel. The product was obtained as a light yellowpowder, 65 mg (61% yield based on the TFA salt precursor); TLCR_(f)=0.27 (DCM-MeOH 5%). [α]₂₃ ^(D)—4.5 (c 0.2, CH₃OH); ¹H (CDCl₃, 400MHz) δ 8.82 (1H, bs), 8.07 (1H, d, J=8 Hz), 7.85 (1H, s), 7.71 (1H, d,J=8.8 Hz), 7.41 (1H, d, J=8.3 Hz), 7.31 (1H, dd, J=8, 4.7 Hz), 6.88 (1H,d, J=8.64 Hz), 6.71 (1H, m), 4.73 (1H, dd, J=9.6, 6.8 Hz), 4.02 (1H, m),3.95 (1H, m), 3.78 (1H, dd, J=8.7, 1.6 Hz), 3.71-3.48 (3H, m), 3.33-3.28(2H, m), 3.27 (3H, s), 3.22 (3H, s), 3.02 (2H, t, J=7.6 Hz), 2.97 (3H,bs), 2.40 (1H, d, J=7.1 Hz), 2.34-2.26 (2H, m), 2.20 (6H, s), 2.09-1.71(5H, m), 1.62 (3H, m), 1.30 (1H, m), 1.16 (3H, d, J=7.2 Hz), 0.99-0.84(16H, m), 0.76 (3H, t, J=7.9 Hz); ¹³C-NMR (CDCl₃, 100 MHz) 174.2, 171.7,170.3, 170.0, 150.3, 148.3, 141.0, 135.7, 128.7, 128.0, 127.8, 126.8,120.6, 81.7, 78.3, 76.4, 61.8, 60.5, 59.3, 58.2, 57.9, 53.7, 53.4, 47.6,46.7, 44.4, 42.8, 42.7, 40.2, 37.4, 35.7, 33.0, 30.9, 27.6, 25.7, 24.8,20.1, 17.7 (×2), 15.8, 14.8, 10.7; (+)HRAPCIMS m/z 753.5292 [M+H]⁺(calcd for C₄₂H₆₉N₆O₆, 753.5279).

Example 5. Quinstatin 8 8-(1′-Boc-Dap-2′-ethyl)-quinoline

8-(1′-amino-2′-ethyl)-quinoline (0.1 mL, 0.7 mmol) was added to asolution of Boc-Dap⁶ (0.2 g, 0.7 mmol) in anhydrous DCM (3 mL) and thereaction mixture was stirred at 0° C. Next, TEA (0.4 mL, 2.8 mmol, 4equiv.) and DEPC (0.3 mL, 0.2 g, 2.0 mmol, 3 equiv.) were added. Thereaction mixture was stirred at 0° C. for 6 h, and overnight at roomtemperature for 16 h, then concentrated to a dark brown solid which wasseparated by sg flash chromatography using DCM-MeOH 5%; Column size (33cm×2 cm). Fractions were combined according to TLC data. The product wasobtained as a brown oil (quantitative); ¹H (400 MHz, CDCl₃) δ 8.83 (1H,dd, J=4.3, 1.7 Hz), 8.08 (1H, m), 7.63 (1H, m), 7.52 (1H, d, J=6.8 Hz),7.39 (1H, t, J=7.6 Hz), 7.33 (1H, m), 6.90 (1H, m), 3.70 (1H, m), 3.57(3H, m), 3.46-3.32 (3H, m), 3.24 (3H, s, OCH₃), 3.07 (1H, m), 2.21-1.99(1H, m), 1.66 (2H, m), 1.53-1.22 (11H, m), 1.14 (1H, m), 1.02 (2H, m);HRMS (APCI⁺) 442.2703 (M+H)⁺ calcd. for C₂₅H₃₆N₃O₄, 442.2706,

8-(1′-Dap-amino-2′-ethyl)-quinoline Trifluoroacetate salt

A solution of the preceding amide (0.3 g, 0.68 mmol) in dry DCM (10 mL)was stirred at 0° C. under N₂. TFA (1.0 mL, 1.49 g, 13 mmol, 19 equiv.)was added and the reaction mixture stirred at 0° C. for 3 h andmonitored by TLC, DCM-MeOH 5%. The solvent was removed under reducedpressure and using toluene as an azeotrope, then dried using high vacuumfor 16 h to yield a brown oil used without further purification in thenext step.

8-(1′-Dov-Val-Dil-Dap-2′-ethyl)-quinoline (Quinstatin 8)

The preceding TFA salt (0.3 g, 0.66 mmol) was dissolved in dry DCM (10mL) and stirred at 0° C. Dov-Val-Dil-TFA⁷ (0.37 g, 0.66 mmol, 1 equiv.)was added followed by TEA (0.5 mL, 3.6 mmol, 5 equiv.) and DEPC (0.11mL, 7.15 mmol, 11 equiv.). The reaction mixture was stirred under N₂ for4 h at 0° C. Solvent was removed under reduced pressure and the residuewas dried under high vacuum. Chromatographic separation was achievedusing flash sg, eluting with DCM-MeOH 5%; Column size: 20 cm×4 cm; togive the desired product as a light yellow powder 320 mg; TLC R_(f)=0.4(DCM-MeOH 5%); [α]₂₃ ^(D)—12.2 (c 0.5, CH₃OH); ¹H (CDCl₃, 400 MHz),Doubling of signals in the proton and carbon spectra indicating thepresence of two isomers, a pattern observed in dolastatin 10 anddiscovered to be due to conformational isomers arising from cis-transisomerism at the Dil-Dap bond,^(3b) δ 8.89 (1H, m), 8.16, 8.12 (1H, d,J=8 Hz), 7.70, 7.66 (1H, d, J=8 Hz), 7.57 (1H, d, J=6.8 Hz), 7.48, 7.44(1H, d, J=8 Hz), 7.46-7.36 (1H, m), 6.96 (1H, bt, J=4.4 Hz), 6.86 (1H,d, J=8.8 Hz), 4.85, 4.74 (1H, dd, J=6.4, 9.6 Hz), 4.0 (1H, m), 3.80 (1H,dd, J=7.6, 2.9 Hz), 3.72-3.56 (3H, m), 3.53-3.35 (4H, m), 3.30 (3H, s,OCH₃), 3.27 (3H, s, OCH₃), 3.26, 3.25 (3H, s), 3.08 (1H, s), 2.96 (2H,bs), 2.50-2.32 (2H, m), 2.23-2.20 (6H, bs), 2.08-1.77 (5H, m), 1.69-1.51(3H, m), 1.31 (1H, m), 1.10 (2H, d, J=7 Hz), 1.04 (1H, m), 1.0-0.83(15H, m), 0.77 (3H, t, J=7.6 Hz); ¹³C-NMR (CDCl₃, 100 MHz) 173.9, 171.6,170.3, 170.0, 149.4, 147.2, 138.4, 136.9, 136.6, 130.4, 130.1, 128.5,128.4, 127.0, 126.8, 126.7, 126.5, 121.1, 120.9, 86.1, 82.1, 76.4, 61.6,60.3, 59.0, 58.9, 58.0, 57.9, 53.7, 47.5, 46.4, 44.1, 42.9, 41.7, 41.1,37.5, 35.7, 33.1, 30.9, 30.86, 30.7, 27.6, 25.7, 25.6, 25.0, 24.7, 23.4,20.2, 19.8, 17.8, 17.7, 15.2, 14.1, 10.8, 10.2; (+)HRAPCIMS m/z 753.5285(M+H)⁺ (calcd for C₄₂H₆₉N₆O₆, 753.5279).

Example 6. Inhibition of Human Cancer Cell Growth

Cancer Cell Line Procedures.

Inhibition of human cancer cell growth was assessed using the NationalCancer Institute's standard sulforhodamine B assay as previouslydescribed.¹⁰ In summary, cells in a 5% fetal bovine serum/RPMI1640medium were inoculated in 96-well plates and incubated for 24 hrs. Next,serial dilutions of the compounds were added. After 48 h, the plateswere fixed with trichloroacetic acid, stained with sulforhodamine B, andread with an automated microplate reader. A growth inhibition of 50%(GI₅₀ or the drug concentration causing a 50% reduction in the netprotein increase) was calculated from optical density data withImmunosoft® software.

TABLE 1 Human Cancer Cell Lines (GI₅₀ μg/mL [nM]), Growth InhibitionResults from Comparison Experiments. Cell line^(a) NCI- Compound BXPC-3MCF-7 SF-268 H460 KM20L2 DU-145 Dolastatin 10^(1a) (1) 0.0000400.0000042 0.0000043 0.00018 0.0000080 0.0000052 [0.051] [0.005] [0.006][0.229] [0.010] [0.007] Quinstatin 2 (3) 0.0093 0.00031 0.00023 0.00510.00033 0.00031 [12.35] [0.412] [0.306] [6.777] [0.438] [0.412]Quinstatin 3 0.021 0.00023 0.00029 0.031 0.00040 0.00020 [27.9] [0.31][0.38] [41.2] [0.53] [0.26] Quinstatin 6 (4) 0.0020 0.00040 0.000300.0043 0.00039 0.00030 [2.657] [0.532] [0.399] [5.714] [0.518] [0.399]Quinstatin 7 0.0031 0.000030 0.000024 0.011 0.000041 0.000030 [4.12][0.039] [0.032] [14.61] [0.054] [0.040] Quinstatin 8 0.00050 0.0000400.000023 0.00090 0.000040 0.000040 [0.425] [0.053] [0.030] [1.19][0.053] [0.053] Auristatin E^(3a)(2a) >0.001 0.00017 0.00036 0.000390.00036 0.00031 [>1.37] [0.232] [0.492] [0.533] [0.492] [0.424]Desmethylauristatin >0.001 0.00029 0.00031 0.00049 0.00043 0.00030E⁵(2b) [>1.39] [0.404] [0.432] [0.683] [0.599] [0.418] ^(a)Cancer celllines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung(NCI-H460); colon (KM20L2); prostate (DU-145).

Table 1 summarizes a series of cancer cell line growth inhibitionexperiments performed in parallel with very revealing and importantresults. Very noteworthy is the usual cancer biology equivalence ofauristatin E and desmethyl-auristatin E as well as the ten to onehundred times increase in inhibition provided by Quinstatin 2 ascompared to auristatin AQ.^(3a) In one of these experiments Quinstatin 8delivered inhibitions at the dolastatin 10 level, but not consistently.

REFERENCES

The following references are hereby incorporated by reference in theirentireties:

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While particular materials, formulations, operational sequences, processparameters, and end products have been set forth to describe andexemplify this invention, they are not intended to be limiting. Rather,it should be noted by those ordinarily skilled in the art that thewritten disclosures are exemplary only and that various otheralternatives, adaptations, and modifications may be made within thescope of the present invention. Accordingly, the present invention isnot limited to the specific embodiments illustrated herein, but islimited only by the following claims.

What is claimed is:
 1. A compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein R₁ is selectedfrom H, (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, a ProtectingGroup or a Linker Unit; R₂ is selected from H, (C₁-C₆) alkyl, (C₂-C₆)alkenyl or (C₂-C₆) alkynyl; R₃ is H or (C₁-C₆) alkyl; and X is abicyclic heterocyclic ring system selected from quinolinyl,isoquinolinyl, 1,5-naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, 2,7-naphthyridinyl, 1,8-naphthyridinyl, 2,6-naphthyridinyl,phthalazinyl, 2H-chromenyl, 1H-1,5-benzodiazepinyl, 1,2,3-benzotriazinyland 2,5-benzodiazocinyl, wherein the bicyclic heterocyclic ring systemis unsubstituted or substituted with 1, 2 or 3 substituent(s) selectedfrom (C₁-C₆) alkoxy, methylene dioxy, hydroxyl, O-Protecting Group andO-Linker Unit.
 2. The compound of claim 1, wherein the compound hasformula (Ia):


3. The compound of claim 1, wherein the compound has formula (Ib):


4. The compound of claim 1, wherein the compound has formula (Ic):


5. The compound of claim 1, wherein the compound has formula (Id):


6. The compound of claim 1, wherein the compound has formula (Ie):


7. The compound of claim 1, wherein the compound has formula (If):


8. The compound of claim 1, wherein the compound has formula (Ig):


9. The compound of claim 1, wherein R₁ is H, (C₁-C₆) alkyl or a LinkerUnit.
 10. The compound of claim 1, wherein R₂ is H or (C₁-C₆) alkyl. 11.The compound of claim 1, wherein the Linker Unit has formula:A_(a)W_(w)Y_(y), wherein A_(a) is maleimidocaproyl, W_(w) isValine-Citrulline and Y_(y) is p-aminobenzyloxycarbonyl.
 12. A compoundselected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 13. A pharmaceuticalcomposition comprising a compound of claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier. 14.An article of manufacture comprising the compound of claim 1, acontainer, and a package insert or label indicating that the compoundcan be used to treat cancer characterized by the overexpression of atleast one tumor-associated antigen.
 15. A method for killing orinhibiting the proliferation of tumor cells or cancer cells comprisingtreating the tumor cells or cancer cells with a compound of claim 1, ora pharmaceutical composition of claim 13, in an amount effective to killor inhibit the proliferation of the tumor cells or cancer cells.
 16. Amethod for treating cancer in a patient in need thereof comprisingadministering to the patient a compound of claim 1, or a pharmaceuticalcomposition of claim 13, wherein the compound or pharmaceuticalcomposition is administered in an amount effective to treat cancer. 17.A method of determining inhibition of cellular proliferation by acompound, comprising contacting cells in a cell culture medium with thecompound of claim 1 and measuring the cytotoxic activity of thecompound, whereby proliferation of the cells is inhibited.
 18. A methodof inhibiting the growth of tumor cells that overexpress atumor-associated antigen comprising administering to a patient thecompound of claim 1 conjugated to an antibody that is specific for saidtumor-associated antigen, and optionally a second therapeutic agentwherein the compound and the second therapeutic agent are eachadministered in amounts effective to inhibit the growth of tumor cellsin the patient.